Architecture of the Tn7 Posttransposition Complex: An Elaborate Nucleoprotein Structure

https://doi.org/10.1016/j.jmb.2010.06.003Get rights and content

Abstract

Four transposition proteins encoded by the bacterial transposon Tn7, TnsA, TnsB, TnsC, and TnsD, mediate its site- and orientation-specific insertion into the chromosomal site attTn7. To establish which Tns proteins are actually present in the transpososome that executes DNA breakage and joining, we have determined the proteins present in the nucleoprotein product of transposition, the posttransposition complex (PTC), using fluorescently labeled Tns proteins. All four required Tns proteins are present in the PTC in which we also find that the Tn7 ends are paired by protein–protein contacts between Tns proteins bound to the ends. Quantification of the relative amounts of the fluorescent Tns proteins in the PTC indicates that oligomers of TnsA, TnsB, and TnsC mediate Tn7 transposition. High-resolution DNA footprinting of the DNA product of transposition attTn7∷Tn7 revealed that about 350 bp of DNA on the transposon ends and on attTn7 contact the Tns proteins. All seven binding sites for TnsB, the component of the transposase that specifically binds the ends and mediates 3′ end breakage and joining, are occupied in the PTC. However, the protection pattern of the sites closest to the Tn7 ends in the PTC are different from that observed with TnsB alone, likely reflecting the pairing of the ends and their interaction with the target nucleoprotein complex necessary for activation of the breakage and joining steps. We also observe extensive protection of the attTn7 sequences in the PTC and that alternative DNA structures in substrate attTn7 that are imposed by TnsD are maintained in the PTC.

Introduction

Transposable elements are discrete DNA segments that can move between nonhomologous positions within genomes. They are present in virtually every genome that has been examined and in some instances form a substantial fraction of the genome; for example, at least 45% of the human genome is derived from transposable elements.1 Transposable elements also have a substantial effect on bacterial genomes.2

Element-encoded transposases bind specifically to transposon ends and mediate the catalytic steps in transposition, cleaving the phosphodiester bonds that link the transposon to the donor site and joining the transposon ends to the insertion site.3, 4, 5 The transposase often acts in concert with other element- and/or host-encoded proteins to assemble the nucleoprotein complexes called transpososomes that mediate key steps in transposition, such as pairing of the transposon ends and selection of a target site.6, 7, 8 The proper nucleoprotein architecture of these complexes is central to the activation and coordination of the DNA breakage and joining events that underlie transposition.

Tn7 transposition occurs by a “cut-and-paste” mechanism in which the transposon is excised from the donor site and integrated into the target site.9, 10 This process is tightly regulated by the recognition of the target site and assembly of a TnsABC + D transpososome: no excision of Tn7 from the donor site occurs unless attTn7 and all the Tns proteins are present.11 Using DNA cross-linking, we have previously identified nucleoprotein complexes containing both a donor DNA from which Tn7 has not yet been excised and a target DNA whose formation requires all the Tns proteins.12 The architecture of the transpososome that mediates DNA breakage and joining must result in the juxtaposition of three DNA sites, the left (Tn7.L) and right (Tn7.R) ends of the transposon and a target DNA. We show here that all the Tns proteins required for insertion into attTn7 are still present in the nucleoprotein complex in which breakage and joining have occurred.

Tn7's transposition machinery is especially elaborate.9, 10 Unlike simpler systems that utilize one or two transposition proteins,13 four Tn7-encoded proteins, TnsA, TnsB, TnsC, and TnsD, are required to insert Tn7 into a specific site in bacterial chromosomes called attTn7.11 Tn7.L and Tn7.R both contain multiple specific binding sites for the transposase subunit TnsB, but these sites are differently positioned on each end, making the ends structurally asymmetric.14, 15 The ends of Tn7 are also functionally asymmetric15, 16 Tn7 elements with two Tn7.Rs transpose, whereas those with two Tn7.Ls do not. Furthermore, Tn7 insertion into attTn7 downstream of the highly conserved glmS gene is orientation specific as well as site specific.17

attTn7 is chosen as a specific site for Tn7 insertion by the binding of TnsD to a particular sequence at the end of the highly conserved glmS gene.11, 18, 19 The exceptional conservation of the TnsD binding site within the region of the glmS encoding the GlmS active site provides a specific insertion site for Tn7 in virtually all prokaryotic genomes that have been examined.20 Similar sequences in the human glmS homologs provide specific sites for the high-frequency insertion of Tn7 in vitro.19, 21

TnsA and TnsB form the transposase that carries out Tn7 DNA breakage and joining.22, 23, 24 The TnsAB transposase is not, however, constitutively active: transposase activity is controlled by interaction of TnsAB with TnsCD bound to attTn7.11 Selection of attTn7 as the target DNA is initiated by TnsD binding to its 30 -bp binding site, which generates distortions in attTn7;11, 18 TnsC is recruited to this distorted DNA.25 The interaction of TnsC with TnsD–attTn7 is ATP dependent11 and leads to the regulation of transposase activity via TnsC interactions with both TnsA and TnsB.12, 2628

At what stages of transposition are the various Tns proteins required? For example, does TnsD simply serve as an “assembly” factor that dissociates from the rest of the transposition machinery once TnsC is loaded onto the attTn7 target DNA, as is seen for the targeting protein UvrA within the nucleotide excision repair pathway.29 We report here our analysis of the architecture of the Tn7 transpososome in which transposition has occurred, the posttransposition complex (PTC). We demonstrate that the PTC is an elaborate nucleoprotein complex in which the Tn7 ends are paired and contains all four Tns proteins that are required to execute transposition, TnsABCD, in addition to the DNA product of transposition, the simple insertion DNA attTn7∷Tn7. We also characterize the extensive Tns protein–DNA contacts within the PTC.

After Tns-mediated DNA breakage and joining, conversion of the attTn7∷Tn7 transposition product to intact duplex DNA requires repair by host proteins of the single-strand gaps that flank the 5′ ends of the newly inserted transposon. These single-strand gaps result from the attack of the transposon's 3′ ends on staggered positions in the target DNA. We find that such repair cannot occur on the PTC in vitro. Thus, host proteins are also likely involved in disassembly of the PTC to remove the Tns proteins such that host DNA repair machinery can access the newly inserted transposon. Host proteins have been shown to remodel the Mu transpososome after transposition to allow DNA replication and repair.30, 31, 32

Section snippets

Tn7 PTCs can be visualized by atomic force microscopy

Using a supercoiled Tn7 donor plasmid and a small linear attTn7 target fragment as DNA substrates, we carried out transposition reactions and then used multiple cycles of size filtration and washing to isolate the stable PTCs from unincorporated Tns proteins (Fig. 1a). In contrast to pretransposition complexes that are detected only upon cross-linking,12 the PTC is stable in the absence of cross-linking, indicating that an increase in stability of Tns–DNA complexes accompanies Tn7

Tn7 transpososomes are elaborate and stable nucleoprotein complexes

Successful transposition requires specific recognition and juxtaposition of the transposon ends and the target DNA, and careful coordination of the DNA breakage and joining events, avoiding nonproductive DNA breaks at the transposon ends and promoting equivalent joining of the transposon ends to the target DNA. These reactions occur in nucleoprotein complexes called transpososomes.6, 8 We have defined the components and architecture of the transpososome product of Tn7 transposition, the PTC,

Tns proteins

The tnsA, tnsB, tnsC, and tnsD genes were cloned into pCYB1 [New England Biolabs (NEB)] to express Tns–intein–chitin binding domain fusion proteins for affinity purification on chitin beads. One-liter cultures of CAG45623 containing Tns–intein–chitin binding domain fusion proteins in the pCYB1 vector were grown at 30 °C to an OD of 0.5 and cooled on ice for 20 min before induction with 400 μM IPTG for 18 h at 16 °C. Cells were harvested by centrifugation and resuspended in 10 ml lysis buffer

Acknowledgements

We thank Robert Sarnovsky for cloning of TnsC into pCYB1; Prasad Kuduvalli, Gregory McKenzie, Chuck Merryman, and Rupak Mitra for helpful discussions; Jan Hoh for assistance with AFM; and Iva Ivanovska, Helen McComas, and Patti Kodeck for assistance with manuscript preparation. J.W.H. and N.L.C. conceived of and designed the experiments. J.W.H. performed the experiments and J.W.H. and N.L.C. wrote the paper. This work was supported by NIH grants P01 CA16519 and RO1 GM076425 to N.L.C. and NIH

References (51)

  • NakaiH. et al.

    Disassembly of the bacteriophage Mu transposase for the initiation of Mu DNA replication

    J. Biol. Chem.

    (1995)
  • YanagiharaK. et al.

    Progressive structural transitions within Mu transpositional complexes

    Mol. Cell

    (2003)
  • LembergK.M. et al.

    The dynamic Mu transpososome: MuB activation prevents disintegration

    J. Mol. Biol.

    (2007)
  • KennedyA. et al.

    Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity

    Cell

    (2000)
  • BollandS. et al.

    The three chemical steps of Tn10/IS10 transposition involve repeated utilization of a single active site

    Cell

    (1996)
  • StellwagenA. et al.

    Mobile DNA elements: controlling transposition with ATP-dependent molecular switches

    Trends Biochem. Sci.

    (1998)
  • LanderE. et al.

    Initial sequencing and analysis of the human genome

    Nature

    (2001)
  • De PalmenaerD. et al.

    IS4 family goes genomic

    BMC Evol. Biol.

    (2008)
  • MizuuchiK. et al.

    Chemical mechanisms for mobilizing DNA

  • HarenL. et al.

    Integrating DNA: transposases and retroviral integrases

    Annu. Rev. Microbiol.

    (1999)
  • HickmanA. et al.

    Integrating prokaryotes and eukaryotes: DNA transposases in light of structure

    Crit. Rev. Biochem. Mol. Biol.

    (2010)
  • CraigN.

    Tn7

  • PetersJ. et al.

    Tn7: smarter than we thought

    Nat. Rev. Mol. Cell. Biol.

    (2001)
  • SkeldingZ. et al.

    Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation

    EMBO J.

    (2002)
  • CraigN. et al.

    Mobile DNA II

    (2002)
  • Cited by (16)

    • Structural basis of transposon end recognition explains central features of Tn7 transposition systems

      2022, Molecular Cell
      Citation Excerpt :

      This is apparent from the fact that the DNA around the TnsA cut site is not accessible in our model of the STC. Moreover, it has been shown that the pre-transposition complex is less stable than the post-transposition complex (Holder and Craig, 2010; Skelding, 2002) and that transposon end protection changes between the TnsB complex alone and the post-transposition complex (Holder and Craig, 2010). Specific interactions between TnsA and TnsB likely manifest themselves in our finding that multiple substitutions in TnsB have a modest effect on DNA binding as shown by NanoLuc but completely abolish the transposition.

    • Moving DNA around: DNA transposition and retroviral integration

      2011, Current Opinion in Structural Biology
      Citation Excerpt :

      Although PFV integrase will perform coordinated integration of a pair of short viral DNA ends such as those in the crystal structure, HIV integrase requires much longer DNA substrates to form a proper intasome [36], suggesting that the outer subunits, and maybe many other weakly interacting ones as well, might form a complex network of stabilizing protein–protein and protein–DNA interactions. Tn7 and bacteriophage Mu transposases are examples of systems that include specific binding sites for subunits not needed for the chemical steps of transposition [37,38]. In these cases the binding site arrays differ between the left and right element ends, and the ‘extra’ subunits may somehow aid in pairing one left with one right end, as well as in stabilizing the full complex.

    • The emerging diversity of transpososome architectures

      2012, Quarterly Reviews of Biophysics
    View all citing articles on Scopus
    1

    Present address: J. W. Holder, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

    View full text